While fatty acids are essential components of membrane structure and energy storage, their functions in cellular signaling, protein activation, and regulation of inflammatory, immune and other stress responses are equally important. The long-range goal of this project is to more broadly characterize the metabolism of acyl- CoAs and to identify the consequences of its failure in humans. Specifically, this renewal application will further examine the metabolism of branched chain and other unusual fatty acids. In humans, the acyl-CoA dehydrogenase (ACD) family consists of 11 evolutionarily conserved flavoenzymes involved in either branched chain amino acid metabolism or fatty acid 2-oxidation. Genetic deficiencies that cause significant morbidity and mortality have been identified in nine of the ACDs. We identified two new ACDs (ACD10 and 11) that are the most evolutionarily conserved members of this family. They have a more complicated gene structure and more limited tissue distribution than the other ACDs. They also appear to use less abundant, unusual substrates. This renewal application has three specific aims. Specific Aim 1 is to characterize the role of ACD10 and 11 in human metabolism a. Specific aim 1a is to characterize the enzymatic properties of the two major products of the ACD10 and 11 loci. In contrast to other ACDs, the ACD10 and 11 genes include an extra APH domain at the N-terminus. I hypothesize that in humans these genes will support expression of at least two major proteins, one containing both domains and another consisting of just the ACD domain, that will differ in their subcellular localization. I predict that the ACD10 and 11 proteins containing only the ACD domain will be located in mitochondrial. Specific aim 1b is to characterize the substrate specificity of the two primary products of ACD10 and 11. I hypothesize that full length ACD11 degrades aromatic butyrate derivatives in peroxisomes, while the shorter form oxidizes isobranched chain and straight chain acyl-CoA substrates with 1- carbon backbones of more than 18 carbons in mitochondria. Specific Aim 2 is to characterize an ACD11 deficient mouse. Specific Aim 2a is to characterize the biochemical phenotype of the ACD11 deficient mouse. Specific Aim 2b is to characterize the clinical phenotype of the ADC11 deficient mouse. Specific Aim 3 is to identify and characterize patients with deficiencies in degrading branched chain acyl-CoAs. Specific Aim 3a is to identify patients with a deficiency of ACD10 and 11. Specific Aim 3b is to expand the characterization of SBCAD and IBD deficiency to understand their clinical and mutation spectrums.